Major areas targeted for genetic improvement in plants include herbicide, insect, and disease resistance. Herbicide resistance can be obtained by genetically engineering plants to respond to toxic chemicals. Many herbicides act by inhibiting a key plant enzyme or protein necessary for growth. For example, the herbicide glyphosate destroys plants by inhibiting the activity of an enzyme necessary for synthesizing aromatic amino acids. Some bacteria contain enzymes that confer resistance to herbicides. A gene encoding a herbicide resistant enzyme from bacteria may be cloned, modified for expression in plants, and inserted crop plant genomes. When sprayed with such herbicide, plants containing the bacterial gene grow as well as unsprayed control plants. For example, the herbicide glyphosate acts by inhibiting the enzyme 5-enolpyruvyl-3-phosphoshikimate synthase (EPSPS). Glyphosate tolerant plants have been produced by inserting EPSPS into the plant genome (U.S. Pat. No. 5,312,910).
Genetic engineering has also been used to protect plants from bacterial, fungal, and virus infection (D'Maris et. al., Trends in Microbiol. 6:54–61 (1998)). It has been shown that transgenic plants that express the coat protein of a virus become resistant to infection by that virus. Although the mechanism of resistance is unknown, the presence of viral coat protein in plant cells can interfere with the uncoating of viral particles containing that coat protein, thereby interrupting the virus replication cycle. Viral replicase and movement protein genes have also been used to confer resistance to viral pathogens. For example, mutated forms of viral genes may be added to the plant genome and expressed in plants for protection against viruses. U.S. Pat. No. 5,773,701 issued to Braun et al., describes the insertion of a potato virus X (PVX) replicase gene into a plant to confer resistance against PVX infection when the PVX replicase gene is sufficiently expressed. PCT publication WO 98/59046 describes the incorporation of β-glucanase gene in transgenic plants for increased resistance to fungal infection.
Proteins conferring insect resistance have also been genetically introduced into plants. Bt-toxins from Bacillus thuringiensis are toxic to moth, fly larvae, beetle, and mosquitoes. A Bt-toxin gene may be cloned into a plasmid vector under control of a chloroplast rRNA promoter and transferred into plant chloroplasts by particle bombardment. Plants that sufficiently express this protein are toxic to insect larvae.
The approach of using transgenic plants in acquiring herbicide, insect and disease resistance has many drawbacks. Because foreign genes are permanently inserted into the genome, development of transgenic plants can be costly and their durability could become a concern. In many cases, the targets of herbicides or pathogens are unknown and indirect. Therefore, finding suitable herbicide-specific or pathogen-specific targets is difficult.
The present invention features a method of using transient plant viral expression vectors to express enzymes or proteins to confer resistance to herbicides, pests, and diseases in plant hosts. Viruses are a unique class of infectious agents whose distinctive features are their simple organization and their mechanism of replication. Their hosts include a wide variety of plants. A complete viral particle, or virion, may be regarded mainly as a block of genetic material (either DNA or RNA) capable of autonomous replication, surrounded by a protein coat and sometimes by an additional membranous envelope. The coat protects the virus from the environment and serves as a vehicle for transmission from one host cell to another.
Virus-based expression systems, in which the foreign mRNA is greatly amplified by virus replication, can produce very high levels of proteins in leaves and other tissues. Viral vector produced protein can also be directed to specific subcellular locations, such as endomembrane, cytosol, or organelles, or it can be attached to macromolecules, such as virions. In addition, virus vectors are ideal for shuttling libraries of sequence variants throughout plant hosts for selection of resistance to herbicides, pests, and diseases.